Abstract Hepatitis C virus (HCV) is a major cause of liver disease, with about 130 million people infected worldwide. No vaccine has been developed, and the only treatment available is a combination of pegylated interferon alpha and ribavirin. Progress in developing novel direct-acting treatments has been greatly hampered by the dearth of novel target proteins. We have developed high-throughput screening assays based on the inhibition of dimerization of HCV core, the viral capsid protein, which is essential for the assembly of the viral particle. These assays, now adopted by the pharmaceutical industry, have allowed us to identify novel inhibitors of HCV production. A validated hit of a screen based on our core-core assays was used as a molecular probe to study the interaction of core with the NS3 helicase of HCV. We will develop new transfer of energy assays to identify high-affinity inhibitors of the core-NS3 helicase interaction. The resulting compounds will constitute novel molecular probes for understanding how HCV non structural proteins participate in the assembly of the viral particle. These inhibitors may also serve as the basis of drugs to be combined with other anti-viral agents to develop treatments that will be more generally applicable than the current interferon-based unsatisfactory regimen. PUBLIC HEALTH RELEVANCE: Narrative Hepatitis C affects 130 million individuals worldwide, 3 million of which live in the US, and the only treatment, a combination of interferon and ribavirin, is effective for less than half of the patients. We will develop a specific transfer-of energy-based assay screening assay for potent molecular probes of the virus, based on inhibition of interaction between core, the capsid protein essential for assembly of the viral particle, and NS3 helicase, a HCV protein essential for viral replication. These compounds may lead to the development of orally available drugs for combination treatment of Hepatitis C.